Project Summary/Abstract Microbial communities exert major impacts on animal biology and human health, and disruption of these communities is associated with multiple disease states. To date, the field of microbiome research has been dominated by surveys of microbial community compositions and by analyses correlating composition with host phenotypes. But there have been few attempts to directly link the specific, causal processes that determine colonization dynamics and success of host-associated bacteria, and how these interactions ultimately affect hosts. The proposed research plan is motivated by the need for experimental systems to identify the mechanisms that control the composition and consequences of host-associated bacterial communities. This work focuses on two model systems that provide complementary approaches to examining host-associated communities, and that offer new opportunities to identify the mechanisms underlying host colonization. The honey bee and its specialized gut microbiota provides an exceptional model for multispecies gut communities as it shares many features with the human gut microbiota. In both human and bee guts, a stable, healthy community bestows ?colonization resistance?, the exclusion of foreign microorganisms; in both systems, disruption can result in dysbiosis and expansion of atypical communities, including enteric pathogens. The human system is highly complex and not amenable to experiments, but, for the bee gut, we are able to culture isolates of all component bacterial species and to introduce these to microbiota-free hosts to establish defined communities. We have already developed genetic tools for experimental manipulation of the dominant species. One set of experiments will identify the direct host-bacterial interactions that determine colonization success or failure of particular strains that vary in ability to colonize honey bees. Existing results from a mutagenesis screen indicate that features of the outer cell envelope play essential roles during host colonization, and we will use new genetic tools to determine which of these factors are key to acceptance by hosts. In addition to elucidating the mechanisms that enable specific bacterial strains to mono-colonize specific hosts, a second set of experiments will investigate how the interactions between microbial strains, which range from metabolic co-dependency to direct toxin-mediated antagonism, determine community membership. The pea aphid and its endosymbionts provide an effective model for how intracellular bacterial associates stably colonize host cells. Our newly devised techniques allow isolation, manipulation and inter-host transfer of endosymbionts. To address how hosts control endosymbiont replication and persistence, we will perform genomic comparisons, biochemical experiments to test effects of host-produced gene products on endosymbiont cells, and physical and structural characterization of endosymbiont outer membrane proteins. Through focus on experimental models that provide tractable examples of host-associated bacteria, this work will illuminate the mechanisms that underlie ability of bacterial symbionts to colonize hosts.